Purpose: Identifying the source of stone formation in recurrent stone formers has always been a big problem. Material and Methods: In this study kidney stones from 52 patients were submitted to direct examination by s...Purpose: Identifying the source of stone formation in recurrent stone formers has always been a big problem. Material and Methods: In this study kidney stones from 52 patients were submitted to direct examination by scanning electronic microscopy (SEM) after manual fracture and 27 calculi were cultured in Eagle’s Minimum Essential Medium (E-MEM) and Brain-Heart Infusion (BHI) for eight weeks at 37°C in 5% CO2 atmosphere. Twenty-seven powdered and demineralized stones were suspended in sterile PBS, filtered through 0.22 m-pore-size sterile filters Millex (Millipore, Massachusetts, USA) and submitted to DNA extraction (Quiagen-Brazil). Platinum PCR SuperMIX (GIBCO-BRL), primers (Invitrogen), and Ultra Pure Water (Advanced Biotechnologies, Columbia, MD) were used for PCR (Polymerase Chain Reaction), which was generally conducted for 30 or 35 cycles with annealing of primers for 40 sec at 55°C and extension for 1 min at 72°C. Results: In 36 out 52 (69%) kidney stones it was detected the presence of biofilm coating the mineral surface of the stone when examined by SEM, consisting of coccoid particles, isolated or clustered, with diameter of 500 nm or less. Eleven out 27 (41%) kidney stone cultures produced white-colored sediment on the bottom of the tubes after eight-week incubation, revealing tiny structures similar to those observed directly by SEM. These structures were similar in size and morphology to spherical apatite particles previously observed in human kidney stones and named as nanobacteria (NB). No PCR products were observed in the samples. Conclusion: We found a strong correlation between renal stones and calcifying nanoparticles (CNP) in this study and these results open a new insight on this area to explore the etiology of stone formation. Whether NB/CNP are truly microorganisms or self-propagating mineral compounds is still controversial and its contribution, if any, in apatite nucleation and crystal growth remains uncertain.展开更多
Objective: Analysis of the effect of nanobacteria on the expression of local biomarkers in patients with rheumatoid arthritis. Methods: Serum samples from 54 patients with rheumatoid arthritis in our hospital were tes...Objective: Analysis of the effect of nanobacteria on the expression of local biomarkers in patients with rheumatoid arthritis. Methods: Serum samples from 54 patients with rheumatoid arthritis in our hospital were tested for nanobacteria, and were divided into three groups based on the concentration of nanobacteria. To compare the differences of main essential elements, serum cytokines, immunochemical indicators and bone metabolic markers in synovial fluid of three groups of patients. Results: Differences in serum calcium levels between the three groups are significant. The differences in IL-1, TNF-α and CRP were significant. The difference of IgM, IgA, anti-CCPP content are significant. CTX-1, RF, ESR content differences are significant. Conclusions: The detection of nanobacteria in joint fluid of patients with rheumatoid arthritis has certain effects on the expression of serum cytokines and immune indexes. The higher the concentration of nanobacteria, the higher the expression of inflammatory and immunological indicators in serum.展开更多
In 1990, nanobacterium was found and named by Kajander.~1 With distinct mineralizing ability, nanobacteria are thought to play a role in extraskeletal calcifying diseases. It have been found in many human tissues, but...In 1990, nanobacterium was found and named by Kajander.~1 With distinct mineralizing ability, nanobacteria are thought to play a role in extraskeletal calcifying diseases. It have been found in many human tissues, but whether they exist in the bile or gallbladder mucosa remains unclear. The present study was undertaken to investigate by ELISA, bacterial culturing, immunohistochemical staining and transmission electron microscopy (TEM), whether nanobacteria exist in serum, bile or gallbladder mucosa of healthy people and patients with cholecystolithiasis.展开更多
目的:研究在纳米细菌诱导实验性大鼠肾结石形成过程中,大鼠肠道草酸降解菌的改变及其与肾结石形成的关系。方法:30只4周龄Wistar雄性大鼠适应性喂养1周后,按照随机数表法分为三组,对照组(NC组):1.2 ml 0.9%氯化钠溶液尾静脉注射+2 ml 0...目的:研究在纳米细菌诱导实验性大鼠肾结石形成过程中,大鼠肠道草酸降解菌的改变及其与肾结石形成的关系。方法:30只4周龄Wistar雄性大鼠适应性喂养1周后,按照随机数表法分为三组,对照组(NC组):1.2 ml 0.9%氯化钠溶液尾静脉注射+2 ml 0.9%氯化钠溶液灌胃;干扰组(NBT组):1.2 ml纳米细菌悬液尾静脉注射+2 ml四环素溶液灌胃;纳米细菌组(NB组):1.2 ml纳米细菌悬液尾静脉注射+2 ml 0.9%氯化钠溶液灌胃。标准饲养8周后处死,取双侧肾脏组织行HE、Pizzolatto’s染色,称取1 g回盲部新鲜粪便并提取DNA,使用qPCR法检测产甲酸草酸杆菌(Oxc)、乳杆菌属细菌(Lac)、双歧杆菌属细菌(Bif)和肠杆菌科细菌(Ent)四种主要肠道草酸降解细菌的基因表达量,留取腹主动脉血液及肾脏组织,流式细胞仪检测淋巴细胞分化情况,WB检测骨桥蛋白(OPN)及Tamm-Horsfall蛋白(THP)两种炎性蛋白在肾脏组织中的表达情况。结果:NC组肾小管管腔内未见明显晶体及炎症细胞浸润,NBT组部分肾小管扩张,肾小管管腔内可见不规则晶体及炎症细胞浸润,NB组肾小管明显扩张,肾小管管腔内可见大量不规则晶体,部分连接成片,可见大量炎症细胞浸润;三组大鼠粪便中均检测到四种肠道草酸降解菌,且各肠道草酸降解菌在三组大鼠盲肠中的占比相似,均为Oxc最高,约占半数,Lac、Ent次之,Bif最少,仅占约1%,三组大鼠粪便中各肠道草酸降解菌占比无统计学差异(均P>0.05),但三组粪便中肠道草酸降解菌的总量有统计学差异,NC组最高、NB组次之、NBT组最低,NC组与其余两组间的差异均有统计学意义(均P<0.01);流式细胞筛选结果提示三组大鼠血液淋巴细胞出现不同程度分化,Pearson相关性分析结果提示OPN与THP联系紧密,增长趋势具有一致性并且THP蛋白与CD4^(+)、CD8^(+)T淋巴细胞也具有强相关性。结论:纳米细菌诱导肾结石形成过程中肠道草酸降解菌总量降低,但各肠道草酸降解菌所占比例无明显改变。展开更多
目的探讨乌梅提取物对纳米细菌(NB)导致的大鼠肾草酸钙结石形成的影响及其机制。方法 30只大鼠随机分为A、B、C、D、E组各6只。A组第1天以生理盐水1 m L尾静脉一次性注射作为正常对照,B、C、D、E组均于第1天以尾静脉一次性注射NB混悬液1...目的探讨乌梅提取物对纳米细菌(NB)导致的大鼠肾草酸钙结石形成的影响及其机制。方法 30只大鼠随机分为A、B、C、D、E组各6只。A组第1天以生理盐水1 m L尾静脉一次性注射作为正常对照,B、C、D、E组均于第1天以尾静脉一次性注射NB混悬液1 m L建立大鼠草酸钙结石模型。A、B组第2天起均以生理盐水2m L/d灌胃,C、D、E组第2天起分别以0.05、0.10、0.15 g/m L乌梅提取物2 m L/d灌胃。各组干预4周后,收集24 h尿液,应用高锰酸钾褪色法测定尿液草酸(Ox)水平,全自动生化仪测定尿液Ca^(2+)水平,柠檬酸试剂盒测定尿液柠檬酸水平,8-羟基脱氧鸟苷(8-OHDG)测试盒测定尿液8-OHDG水平。取左侧肾脏,行HE染色观察肾组织草酸钙结晶形态,行结晶量分级评分。采用硫代巴比妥酸法检测肾组织丙二醛(MDA)水平。结果 B组尿液Ox、Ca^(2+)、8-OHDG水平均高于A组,柠檬酸水平低于A组(P均<0.05);C、D、E组尿液Ox、Ca^(2+)、8-OHDG水平均低于B组,柠檬酸水平均高于B组(P均<0.05)。A组无草酸钙结晶,B组草酸钙结晶明显较多,C、D、E组草酸钙结晶数量较B组明显减少。B组草酸钙结晶量评分高于A组,C、D、E组草酸钙结晶量评分均低于B组(P均<0.05)。B组肾组织MDA水平高于A组,D、E组肾组织MDA水平均低于B组(P均<0.05)。结论乌梅提取物对NB所致的大鼠肾草酸钙结石具有明显的抑制作用,其机制可能与降低大鼠尿液Ox水平,进而减少尿Ca^(2+)排泄、减轻脂质过氧化损伤有关。展开更多
目的构建纳米细菌(NB)致肾结石大鼠模型,观察乌梅提取物对早期肾损伤分子1(KIM-1)和骨桥蛋白(OPN)的影响,分析乌梅提取物对早期肾结石的治疗意义。方法从人上尿路结石分离并培养NB。将54只雄性SD大鼠随机分为空白组、诱石组和治疗组,采...目的构建纳米细菌(NB)致肾结石大鼠模型,观察乌梅提取物对早期肾损伤分子1(KIM-1)和骨桥蛋白(OPN)的影响,分析乌梅提取物对早期肾结石的治疗意义。方法从人上尿路结石分离并培养NB。将54只雄性SD大鼠随机分为空白组、诱石组和治疗组,采取尾静脉注射NB构建肾结石模型,以乌梅提取物干预模型,并在不同时期分批处死大鼠,实时PCR检测肾组织KIM-1 m RNA的表达,ELISA检测尿KIM-1浓度,HE染色观察肾组织结石晶体情况,并用免疫组化方法检测OPN表达。结果诱石组及治疗组肾结石模型大鼠出现肾小管扩张,肾小管结石晶体形成,早期KIM-1和OPN的表达升高,上述变化与NB的注射时间呈正相关,乌梅提取物可拮抗该类变化。结论乌梅提取物可能通过修复肾损伤减少肾结石形成,该机制可能与乌梅提取物对KIM-1、OPN的基因调节有关。展开更多
文摘Purpose: Identifying the source of stone formation in recurrent stone formers has always been a big problem. Material and Methods: In this study kidney stones from 52 patients were submitted to direct examination by scanning electronic microscopy (SEM) after manual fracture and 27 calculi were cultured in Eagle’s Minimum Essential Medium (E-MEM) and Brain-Heart Infusion (BHI) for eight weeks at 37°C in 5% CO2 atmosphere. Twenty-seven powdered and demineralized stones were suspended in sterile PBS, filtered through 0.22 m-pore-size sterile filters Millex (Millipore, Massachusetts, USA) and submitted to DNA extraction (Quiagen-Brazil). Platinum PCR SuperMIX (GIBCO-BRL), primers (Invitrogen), and Ultra Pure Water (Advanced Biotechnologies, Columbia, MD) were used for PCR (Polymerase Chain Reaction), which was generally conducted for 30 or 35 cycles with annealing of primers for 40 sec at 55°C and extension for 1 min at 72°C. Results: In 36 out 52 (69%) kidney stones it was detected the presence of biofilm coating the mineral surface of the stone when examined by SEM, consisting of coccoid particles, isolated or clustered, with diameter of 500 nm or less. Eleven out 27 (41%) kidney stone cultures produced white-colored sediment on the bottom of the tubes after eight-week incubation, revealing tiny structures similar to those observed directly by SEM. These structures were similar in size and morphology to spherical apatite particles previously observed in human kidney stones and named as nanobacteria (NB). No PCR products were observed in the samples. Conclusion: We found a strong correlation between renal stones and calcifying nanoparticles (CNP) in this study and these results open a new insight on this area to explore the etiology of stone formation. Whether NB/CNP are truly microorganisms or self-propagating mineral compounds is still controversial and its contribution, if any, in apatite nucleation and crystal growth remains uncertain.
文摘Objective: Analysis of the effect of nanobacteria on the expression of local biomarkers in patients with rheumatoid arthritis. Methods: Serum samples from 54 patients with rheumatoid arthritis in our hospital were tested for nanobacteria, and were divided into three groups based on the concentration of nanobacteria. To compare the differences of main essential elements, serum cytokines, immunochemical indicators and bone metabolic markers in synovial fluid of three groups of patients. Results: Differences in serum calcium levels between the three groups are significant. The differences in IL-1, TNF-α and CRP were significant. The difference of IgM, IgA, anti-CCPP content are significant. CTX-1, RF, ESR content differences are significant. Conclusions: The detection of nanobacteria in joint fluid of patients with rheumatoid arthritis has certain effects on the expression of serum cytokines and immune indexes. The higher the concentration of nanobacteria, the higher the expression of inflammatory and immunological indicators in serum.
文摘In 1990, nanobacterium was found and named by Kajander.~1 With distinct mineralizing ability, nanobacteria are thought to play a role in extraskeletal calcifying diseases. It have been found in many human tissues, but whether they exist in the bile or gallbladder mucosa remains unclear. The present study was undertaken to investigate by ELISA, bacterial culturing, immunohistochemical staining and transmission electron microscopy (TEM), whether nanobacteria exist in serum, bile or gallbladder mucosa of healthy people and patients with cholecystolithiasis.
文摘目的:研究在纳米细菌诱导实验性大鼠肾结石形成过程中,大鼠肠道草酸降解菌的改变及其与肾结石形成的关系。方法:30只4周龄Wistar雄性大鼠适应性喂养1周后,按照随机数表法分为三组,对照组(NC组):1.2 ml 0.9%氯化钠溶液尾静脉注射+2 ml 0.9%氯化钠溶液灌胃;干扰组(NBT组):1.2 ml纳米细菌悬液尾静脉注射+2 ml四环素溶液灌胃;纳米细菌组(NB组):1.2 ml纳米细菌悬液尾静脉注射+2 ml 0.9%氯化钠溶液灌胃。标准饲养8周后处死,取双侧肾脏组织行HE、Pizzolatto’s染色,称取1 g回盲部新鲜粪便并提取DNA,使用qPCR法检测产甲酸草酸杆菌(Oxc)、乳杆菌属细菌(Lac)、双歧杆菌属细菌(Bif)和肠杆菌科细菌(Ent)四种主要肠道草酸降解细菌的基因表达量,留取腹主动脉血液及肾脏组织,流式细胞仪检测淋巴细胞分化情况,WB检测骨桥蛋白(OPN)及Tamm-Horsfall蛋白(THP)两种炎性蛋白在肾脏组织中的表达情况。结果:NC组肾小管管腔内未见明显晶体及炎症细胞浸润,NBT组部分肾小管扩张,肾小管管腔内可见不规则晶体及炎症细胞浸润,NB组肾小管明显扩张,肾小管管腔内可见大量不规则晶体,部分连接成片,可见大量炎症细胞浸润;三组大鼠粪便中均检测到四种肠道草酸降解菌,且各肠道草酸降解菌在三组大鼠盲肠中的占比相似,均为Oxc最高,约占半数,Lac、Ent次之,Bif最少,仅占约1%,三组大鼠粪便中各肠道草酸降解菌占比无统计学差异(均P>0.05),但三组粪便中肠道草酸降解菌的总量有统计学差异,NC组最高、NB组次之、NBT组最低,NC组与其余两组间的差异均有统计学意义(均P<0.01);流式细胞筛选结果提示三组大鼠血液淋巴细胞出现不同程度分化,Pearson相关性分析结果提示OPN与THP联系紧密,增长趋势具有一致性并且THP蛋白与CD4^(+)、CD8^(+)T淋巴细胞也具有强相关性。结论:纳米细菌诱导肾结石形成过程中肠道草酸降解菌总量降低,但各肠道草酸降解菌所占比例无明显改变。
文摘目的探讨乌梅提取物对纳米细菌(NB)导致的大鼠肾草酸钙结石形成的影响及其机制。方法 30只大鼠随机分为A、B、C、D、E组各6只。A组第1天以生理盐水1 m L尾静脉一次性注射作为正常对照,B、C、D、E组均于第1天以尾静脉一次性注射NB混悬液1 m L建立大鼠草酸钙结石模型。A、B组第2天起均以生理盐水2m L/d灌胃,C、D、E组第2天起分别以0.05、0.10、0.15 g/m L乌梅提取物2 m L/d灌胃。各组干预4周后,收集24 h尿液,应用高锰酸钾褪色法测定尿液草酸(Ox)水平,全自动生化仪测定尿液Ca^(2+)水平,柠檬酸试剂盒测定尿液柠檬酸水平,8-羟基脱氧鸟苷(8-OHDG)测试盒测定尿液8-OHDG水平。取左侧肾脏,行HE染色观察肾组织草酸钙结晶形态,行结晶量分级评分。采用硫代巴比妥酸法检测肾组织丙二醛(MDA)水平。结果 B组尿液Ox、Ca^(2+)、8-OHDG水平均高于A组,柠檬酸水平低于A组(P均<0.05);C、D、E组尿液Ox、Ca^(2+)、8-OHDG水平均低于B组,柠檬酸水平均高于B组(P均<0.05)。A组无草酸钙结晶,B组草酸钙结晶明显较多,C、D、E组草酸钙结晶数量较B组明显减少。B组草酸钙结晶量评分高于A组,C、D、E组草酸钙结晶量评分均低于B组(P均<0.05)。B组肾组织MDA水平高于A组,D、E组肾组织MDA水平均低于B组(P均<0.05)。结论乌梅提取物对NB所致的大鼠肾草酸钙结石具有明显的抑制作用,其机制可能与降低大鼠尿液Ox水平,进而减少尿Ca^(2+)排泄、减轻脂质过氧化损伤有关。
文摘目的构建纳米细菌(NB)致肾结石大鼠模型,观察乌梅提取物对早期肾损伤分子1(KIM-1)和骨桥蛋白(OPN)的影响,分析乌梅提取物对早期肾结石的治疗意义。方法从人上尿路结石分离并培养NB。将54只雄性SD大鼠随机分为空白组、诱石组和治疗组,采取尾静脉注射NB构建肾结石模型,以乌梅提取物干预模型,并在不同时期分批处死大鼠,实时PCR检测肾组织KIM-1 m RNA的表达,ELISA检测尿KIM-1浓度,HE染色观察肾组织结石晶体情况,并用免疫组化方法检测OPN表达。结果诱石组及治疗组肾结石模型大鼠出现肾小管扩张,肾小管结石晶体形成,早期KIM-1和OPN的表达升高,上述变化与NB的注射时间呈正相关,乌梅提取物可拮抗该类变化。结论乌梅提取物可能通过修复肾损伤减少肾结石形成,该机制可能与乌梅提取物对KIM-1、OPN的基因调节有关。
